Brassica oleracea plants with downy mildew resistant curds or heads

ABSTRACT

The present disclosure provides  Brassica oleracea  plants having curds or heads exhibiting increased resistance to downy mildew. Such plants may comprise novel introgressed genomic regions associated with disease resistance from  Brassica oleracea  MYCOCLP. In certain aspects, compositions, including novel polymorphic markers and methods for producing, breeding, identifying, and selecting plants or germplasm with a disease resistance phenotype are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Appl. Ser. No.62/596,601, filed Dec. 8, 2017, the entire disclosure of which isincorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file named “SEMB031US_ST25.txt”,which is 6 kilobytes as measured in Microsoft Windows operating systemand was created on Dec. 6, 2018, is filed electronically herewith andincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture and morespecifically to methods and compositions for producing Brassica oleraceaplants with curds or heads exhibiting improved resistance to Downymildew.

BACKGROUND OF THE INVENTION

Disease resistance is an important trait in agriculture. It isparticularly important for varieties used in the production of foodcrops. In addition to identifying a disease resistance allele, specificmarkers linked to the resistance allele facilitate the introduction ofthe allele into cultivated lines. Marker-assisted selection (MAS) inplant breeding methods has made it possible to select plants based ongenetic markers linked to traits of interest, in this case, diseaseresistance. However, identification of markers for tracking and/orintroducing desirable traits in plants requires significant effort andas such, the markers are often unavailable even if the gene associatedwith the trait has been characterized. The difficulty in identifyingmarkers is also complicated by factors such as polygenic or quantitativeinheritance, epistasis and an often incomplete understanding of thegenetic background underlying expression of a desired phenotype.

SUMMARY OF THE INVENTION

The present disclosure provides a Brassica oleracea plant of acultivated variety, the plant comprising a first introgressed allele ora second introgressed allele on chromosome 3, wherein the firstintrogressed allele or the second introgressed allele confers to a curdor head of the plant increased resistance to downy mildew compared to aplant not comprising the first introgressed allele or the secondintrogressed allele. In certain embodiments the plant comprises a firstintrogressed allele and a second introgressed allele on chromosome 3,wherein the first introgressed allele and the second introgressed alleleconfers to a curd or head of the plant increased resistance to downymildew compared to a plant not comprising the alleles. In someembodiments a sample of seed comprising the first introgressed alleleand the second introgressed allele was deposited under ATCC AccessionNumber PTA-124338. In other embodiments the first introgressed allele isflanked in the genome of the plant by marker locus M19 (SEQ ID NO:2) andmarker locus M20 (SEQ ID NO:3) on chromosome 3. In yet other embodimentsthe second introgressed allele is flanked in the genome of the plant bymarker locus M31 (SEQ ID NO:4) and marker locus M44 (SEQ ID NO:16) onchromosome 3. In additional embodiments the Brassica oleracea plant is abroccoli, cauliflower, sprouting broccoli, Brussels sprouts, whitecabbage, red cabbage, savoy cabbage, curly kale cabbage, turnip cabbageor Portuguese cabbage plant. In particular embodiments the plant ishomozygous for the first introgressed allele or the second introgressedallele.

The present disclosure also provides a seed that produces a Brassicaoleracea plant of a cultivated variety, the plant comprising a firstintrogressed allele or a second introgressed allele on chromosome 3,wherein the first introgressed allele or the second introgressed alleleconfers to a curd or head of the plant increased resistance to downymildew compared to a plant not comprising the first introgressed alleleor the second introgressed allele. In certain embodiments the seedproduces a Brassica oleracea plant of a cultivated variety, the plantcomprising a first introgressed allele and a second introgressed alleleon chromosome 3, wherein the first introgressed allele or the secondintrogressed allele confers to a curd or head of the plant increasedresistance to downy mildew compared to a plant not comprising the firstintrogressed allele and the second introgressed allele.

The present disclosure additionally provides a plant part of a Brassicaoleracea plant of a cultivated variety, the plant comprising a firstintrogressed allele or a second introgressed allele on chromosome 3,wherein the first introgressed allele or the second introgressed alleleconfers to a curd or head of the plant increased resistance to downymildew compared to a plant not comprising the first introgressed alleleor the second introgressed allele. In certain embodiments the plant partis from a Brassica oleracea plant of a cultivated variety, the plantcomprising a first introgressed allele and a second introgressed alleleon chromosome 3, wherein the first introgressed allele or the secondintrogressed allele confers to a curd or head of the plant increasedresistance to downy mildew compared to a plant not comprising the firstintrogressed allele and the second introgressed allele. In particularembodiments the plant part is a cell, a seed, a root, a stem, a leaf, afruit, a flower, a curd, a head or pollen.

The present disclosure further provides an introgression fragmentcomprising a first chromosomal segment on chromosome 3 from Brassicaoleracea MYCOCLP flanked by marker M19 (SEQ ID NO:2) and marker M20 (SEQID NO:3) and a second chromosomal segment on chromosome 3 from Brassicaoleracea MYCOCLP flanked by marker M31 (SEQ ID NO:4) and marker M44 (SEQID NO:16). In certain embodiments the fragment confers increasedresistance to downy mildew. In other embodiments a sample of seedcomprising the first chromosomal segment and the second chromosomalsegment was deposited under ATCC Accession Number PTA-124338.

The present disclosure also provides a method for producing a cultivatedvariety of a Brassica oleracea plant with a curd or head having improvedresistance to downy mildew, comprising introgressing into the plantvariety a first chromosomal segment or a second chromosomal segment fromBrassica oleracea MYCOCLP chromosome 3 that confers improved resistanceto downy mildew relative to a plant lacking the introgression. Incertain embodiments the introgressing comprises crossing a plantcomprising the first or second chromosomal segment with itself or with asecond Brassica oleracea plant of a different genotype to produce one ormore progeny plants, and selecting a progeny plant comprising thechromosomal segment. In other embodiments selecting a progeny plantcomprises detecting at least a first allele flanked by marker M19 (SEQID NO:2) and marker M20 (SEQ ID NO:3) or a second allele flanked bymarker M31 (SEQ ID NO:4) and marker M44 (SEQ ID NO:16). In someembodiments the plant variety is a broccoli, cauliflower, sproutingbroccoli, Brussels sprouts, white cabbage, red cabbage, savoy cabbage,curly kale cabbage, turnip cabbage or Portuguese cabbage plant variety.In additional embodiments the progeny plant is an F2, F3, F4, F5 or F6progeny plant. In particular embodiments the crossing comprisesbackcrossing. In yet other embodiments the backcrossing comprises from2-7 generations of backcrosses, for example 2, 3, 4, 5, 6 or 7generations of backcrosses. In further embodiments the crossingcomprises marker-assisted selection. In yet further embodiments a sampleof seed comprising the first and second chromosomal segment wasdeposited under ATCC Accession Number PTA-124338.

The present disclosure further provides a Brassica oleracea plantproduced by a method for producing a cultivated variety of a Brassicaoleracea plant with a curd or head having improved resistance to downymildew, comprising introgressing into the plant variety a firstchromosomal segment or a second chromosomal segment from Brassicaoleracea MYCOCLP chromosome 3 that confers improved resistance to downymildew relative to a plant lacking the introgression. In addition, thepresent disclosure provides a method of producing food or feedcomprising obtaining a Brassica oleracea plant of a cultivated variety,the plant comprising a first introgressed allele or a secondintrogressed allele on chromosome 3, wherein the first introgressedallele or the second introgressed allele confers to a curd or head ofthe plant increased resistance to downy mildew compared to a plant notcomprising the first introgressed allele or the second introgressedallele, or a Brassica oleracea plant produced by a method for producinga cultivated variety of a Brassica oleracea plant with a curd or headhaving improved resistance to downy mildew, comprising introgressinginto the plant variety a first chromosomal segment or a secondchromosomal segment from Brassica oleracea MYCOCLP chromosome 3 thatconfers improved resistance to downy mildew relative to a plant lackingthe introgression, or a part thereof, and producing the food or feedfrom the plant or part thereof.

The present disclosure also provides a method of selecting a Brassicaoleracea plant exhibiting resistance downy mildew, comprising crossing aBrassica oleracea plant of a cultivated variety, the plant comprising afirst introgressed allele or a second introgressed allele on chromosome3, wherein the first introgressed allele or the second introgressedallele confers to a curd or head of the plant increased resistance todowny mildew compared to a plant not comprising the first introgressedallele or the second introgressed allele, with itself or with a secondBrassica oleracea plant of a different genotype to produce one or moreprogeny plants, and selecting a progeny plant comprising the first orsecond introgressed allele. In certain embodiments selecting the progenyplant comprises identifying a genetic marker genetically linked to thefirst or second introgressed allele. In other embodiments selecting theprogeny plant comprises identifying a genetic marker within orgenetically linked to a genomic region between marker locus M19 (SEQ IDNO:2) and marker locus M20 (SEQ ID NO:3) on chromosome 3, or identifyinga genetic marker within or genetically linked to a genomic regionbetween marker locus M31 (SEQ ID NO:4) and marker locus M44 (SEQ IDNO:16) on chromosome 3. In additional embodiments selecting a progenyplant further comprises detecting at least one polymorphism at a locusselected from the group consisting of marker locus M33 (SEQ ID NO:5),marker locus M34 (SEQ ID NO:6), marker locus M35 (SEQ ID NO:7), markerlocus M36 (SEQ ID NO:8), marker locus M37 (SEQ ID NO:9), marker locusM38 (SEQ ID NO:10), marker locus M39 (SEQ ID NO:11), marker locus M40(SEQ ID NO:12), marker locus M41 (SEQ ID NO:13), marker locus M42 (SEQID NO:14), and marker locus M43 (SEQ ID NO:15). In some embodiments theprogeny plant is an F2, F3, F4, F5 or F6 progeny plant. In furtherembodiments producing the progeny plant comprises backcrossing. Incertain embodiments backcrossing comprises from 2-7 generations ofbackcrossing, for example 2, 3, 4, 5, 6 or 7 generations ofbackcrossing.

The present disclosure also provides a Brassica oleracea plantobtainable by the method of crossing a Brassica oleracea plant of acultivated variety, the plant comprising a first introgressed allele ora second introgressed allele on chromosome 3, wherein the firstintrogressed allele or the second introgressed allele confers to a curdor head of the plant increased resistance to downy mildew compared to aplant not comprising the first introgressed allele or the secondintrogressed allele, with itself or with a second Brassica oleraceaplant of a different genotype to produce one or more progeny plants, andselecting a progeny plant comprising the first or second introgressedallele.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: QTL mapping results for downy mildew resistance in curds. TwoQTL are identified on chromosome 3 that together explain 48.1% of thephenotypic variation.

DETAILED DESCRIPTION

Downy mildew (DM) is caused by an oomycete fungal like pathogen(Hyaloperonospora brassicae, also known as Peronospora parasitica, alsoknown as Hyaloperonospora parasitica). DM is a disease that is foundacross the globe in many brassica species (Brassica oleracea) including,but not limited to, broccoli, cauliflower, cabbage, mustard, radishes,and turnips. DM is more prevalent in regions and seasons with cool, dampweather, high humidity and high levels of dew formation. DM can infectthe plants at any stage of growth but is most frequently identified atthe seedling stage and on mature plants. Infection can occur at thecotyledon stage, seedling production, mature or adult plant stage and inthe curds of cauliflower (Brassica oleracea var. Botrytis) and broccoli(Brassica oleracea var. Italica). Although fungicides may be used tocontrol downy mildew infections, it would be preferable to havevarieties with resistance to limit the damage from DM at each plantstage. In particular, curd resistance is desirable since the applicationof fungicides close to harvest stage is limited due to regulations.

Although certain sources of downy mildew resistance in cauliflower havebeen described in the literature, these sources were generally testedonly for cotyledon resistance and not for adult plant or curdresistance. There is no correlation between resistance at the differentstages of plant development, and resistance at one stage cannot predictresistance at the other stages. In particular, resistance at thecotyledon stage and/or resistance at the adult plant stage cannotpredict resistance in the curd. As a result, one cannot select a sourcefor curd resistance based on foliar tests at either the cotyledon oradult plant stage. Although several sources have been identified thatsuggest resistance at the cotyledon and adult plant stage, it is unknownwhether these same sources would result in resistant curds. Moreover,although certain downy mildew resistance genes have been identified, thegenetic position and molecular markers for these genes have not beenidentified or described.

The experiments to assay for resistance in cauliflower curds requirelong time periods and incur significant costs. The assay requires thatthe plants fully develop in the field and that the plants are maintainedpast the normal harvest time for cauliflower. The trial can require mostof a year to conduct. In addition, the downy mildew pressure oftenvaries between and within seasons. Therefore, in order to obtainmeaningful and reliable results, several experimental trials must beplanted over several different time periods. These types of experimentaltrials are very labor intensive and require access to large fields.

Once a source is identified, marker assisted breeding would improve andincrease the successful introgression of the trait and breeding ofcauliflower with downy mildew resistant curds. A trait linked marker isprimarily useful when the genetics of a trait is relatively simple andthe trait is controlled by a small number of loci, preferably one ortwo.

The present invention represents a significant advance in that itprovides two resistance QTLs that provide increased resistance to downymildew in curds. In addition, trait linked markers are provided whichcan be used to introgress the trait and in the breeding of cauliflowerwith downy mildew resistant curds. The QTLs can be introgressed intoelite lines of cultivated crops of Brassica oleracea. These crops caninclude, but are not limited to, cultivated varieties of broccoli,cauliflower, sprouting broccoli, Brussels sprouts, white cabbage, redcabbage, savoy cabbage, curly kale cabbage, turnip cabbage andPortuguese cabbage.

I. Brassica oleracea Plants

Brassica is a plant genus of the family of brassicaceae (formerlyreferred to as cruciferae). The members of this genus are also known ascabbage or mustard. The genus Brassica comprises a number ofcommercially and agriculturally important species. Of all those speciesBrassica oleracea is the most diverse containing at least ten differentcommercial cultivated varieties, including broccoli, cauliflower,sprouting broccoli, Brussels sprouts, white cabbage, red cabbage, savoycabbage, curly kale cabbage, turnip cabbage and Portuguese cabbage.Breeding between these types is common and easily done because thesetypes, while highly diverse phenotypically, are the same species, whichmeans that a cross between the different types can be made withouthaving to overcome any genetic species barrier. However, significantlinkage drag can still occur for inter-cultivar crosses, especially whencrossing between (genetically) distant cultivars (e.g., a cross betweenwhite cabbage and broccoli or cauliflower). Thus while the absence of aspecies barrier allows crosses to be made between all cultivars, it islikely that linkage drag will be associated with such a cross.

II. Genomic Regions, Alleles, and Polymorphisms Associated with DownyMildew Resistance in Brassica oleracea Plants

The two downy mildew resistance QTLs of the present invention wereidentified on chromosome 3. Each QTL provides resistance to downy mildewby itself and when combined the resistance is additive. The recombinantintrogression fragments were identified using marker assisted breedingtechniques and the introgression fragments generated had sizes of about11 centiMorgans (cM) and 15 cM. The mapping of these chromosomalsegments found that the first QTL for downy mildew resistance is flankedby markers M19 (a SNP change [G/A] at 15,890,285 bp; SEQ ID NO:2) andM20 (a SNP change [T/C] at 10,184,762 bp; SEQ ID NO:3), and the secondQTL is flanked by markers M31 (a SNP change [T/A] at 3,226,172 bp; SEQID NO:4) and M44 (a SNP change [C/T] at 1,221,810 bp; SEQ ID NO:16).Interstitial markers, such as M33, a SNP change [G/A] at 3,178,026 bp(SEQ ID NO:5), M34, a SNP change [T/C] at 2,874,663 bp (SEQ ID NO:6),M35, a SNP change [A/G] at 2,354,342 bp (SEQ ID NO:7), M36, a SNP change[C/T] at 2,168,486 bp (SEQ ID NO:8), M37, a SNP change [A/C] at2,212,440 bp (SEQ ID NO:9), M38, a SNP change [C/A] at 1,973,175 bp (SEQID NO:10), M39, a SNP change [T/G] at 1,391,141 bp (SEQ ID NO:11), M40,a SNP change [A/G] at 1,932,167 bp (SEQ ID NO:12), M41, a SNP change[A/G] at 2,091,771 bp (SEQ ID NO:13), M42, a SNP change [A/G] at1,220,020 bp (SEQ ID NO:14), and M43, a SNP change [A/G] at 1,219,392 bp(SEQ ID NO:15), can be used in addition to the flanking markers toselect for the second resistance QTL on chromosome 3. In certainembodiments, one or both of the flanking markers for the secondresistance QTL are interstitial markers between M31 and M44, such asM33, M34, M35, M36, M37, M38, M39, M40, M41, M42 or M43. The publicgenome positions are based on version 2.1 of the Brassica oleraceagenome (plants.ensembl.org/Brassica_oleracea/Info/index).

One of skill in the art will understand that interval values may varybased on factors such as the reference map that is used, the sequencingcoverage and the assembly software settings. However, such parametersand mapping protocols are known in the art and one of skill in the artcan use the marker sequences provided herein to physically andgenetically anchor the introgressions described herein to any given mapusing such methodology. The novel introgressions of the presentinvention confer unique significantly improved agronomic properties overpreviously disclosed downy mildew resistance introgressions.

Thus in certain embodiments the present disclosure provides Brassicaoleracea plants comprising an introgressed genomic interval flanked bymarkers M19 and M20 or markers M31 and M44. In other embodiments, thepresent disclosure provides Brassica oleracea plants comprising anintrogressed genomic interval flanked by markers M31 and M43, M31 andM42, M31 and M41, M31 and M40, M31 and M39, M31 and M38, M31 and M37,M31 and M36, M31 and M35, M31 and M34, M31 and M33, M33 and M44, M34 andM44, M35 and M44, M36 and M44, M37 and M44, M38 and M44, M39 and M44,M40 and M44, M41 and M44, M42 and M44, M43 and M44, M31 and M44, M33 andM43, M34 and M42, M35 and M41, M36 and M40, or M37 and M39. In furtherembodiments, the present disclosure provides methods of producingBrassica oleracea plants by selecting with any of the above markers.

III. Introgression of Genomic Regions Associated with Disease Resistance

Marker-assisted introgression involves the transfer of a chromosomalregion defined by one or more markers from a first genetic background toa second. Offspring of a cross that contain the introgressed genomicregion can be identified by the combination of markers characteristic ofthe desired introgressed genomic region from a first genetic backgroundand both linked and unlinked markers characteristic of the secondgenetic background.

The present invention provides novel markers for identifying andtracking introgression of one or more of the genomic regions fromBrassica oleracea MYCOCLP (ATCC Accession No. PTA_124338), disclosedherein into cultivated Brassica oleracea lines. The invention furtherprovides markers for identifying and tracking the novel introgressionsdisclosed herein during plant breeding.

Markers within or linked to any of the genomic intervals of the presentinvention can be used in a variety of breeding efforts that includeintrogression of genomic regions associated with disease resistance intoa desired genetic background. For example, a marker within 20 cM, 15 cM,10 cM, 5 cM, 2 cM, or 1 cM of a marker associated with diseaseresistance described herein can be used for marker-assistedintrogression of genomic regions associated with a disease tolerantphenotype.

Brassica oleracea plants comprising one or more introgressed regionsassociated with a desired phenotype wherein at least 10%, 25%, 50%, 75%,90%, or 99% of the remaining genomic sequences carry markerscharacteristic of the germplasm are also provided. Brassica oleraceaplants comprising an introgressed region comprising regions closelylinked to or adjacent to the genomic regions and markers provided hereinand associated with downy mildew disease resistance phenotype are alsoprovided.

IV. Development of Disease Resistant Brassica oleracea Varieties

For most breeding objectives, commercial breeders work within germplasmthat is of a “cultivated variety” or “elite.” This germplasm is easierto breed because it generally performs well when evaluated forhorticultural performance. Numerous Brassica oleracea crop cultivatedvarieties (cultivars) have been developed, including, but not limitedto, broccoli, cauliflower, sprouting broccoli, Brussels sprouts, whitecabbage, red cabbage, savoy cabbage, curly kale cabbage, turnip cabbageand Portuguese cabbage. However, the performance advantage a cultivatedor elite germplasm provides can be offset by a lack of allelicdiversity. Breeders generally accept this tradeoff because progress isfaster when working with cultivated material than when breeding withgenetically diverse sources.

The process of introgressing desirable resistance genes fromnon-cultivated lines into elite cultivated lines while avoiding problemswith linkage drag or low heritability is a long and often arduousprocess. Success in deploying alleles derived from wild relativestherefore strongly depends on minimal or truncated introgressions thatlack detrimental effects and reliable marker assays that replacephenotypic screens. Success is further defined by simplifying geneticsfor key attributes to allow focus on genetic gain for quantitativetraits such as disease resistance. Moreover, the process ofintrogressing genomic regions from non-cultivated lines can be greatlyfacilitated by the availability of informative markers.

One of skill in the art would therefore understand that the alleles,polymorphisms, and markers provided by the invention allow the trackingand introduction of any of the genomic regions identified herein intoany genetic background to which a Brassica oleracea species can becrossed. In addition, the genomic regions associated with diseaseresistance disclosed herein can be introgressed from one genotype toanother and tracked phenotypically or genetically. Thus, Applicants'development of markers for the selection of the disease resistancefacilitates the development of Brassica oleracea plants havingbeneficial phenotypes. For example, plants and seeds can be genotypedusing the markers of the present invention in order to develop varietiescomprising desired disease resistance. Moreover, marker-assistedselection (MAS) allows identification of plants which are homozygous orheterozygous the desired introgression.

Meiotic recombination is essential for plant breeding because it enablesthe transfer of favorable alleles across genetic backgrounds, theremoval of deleterious genomic fragments, and pyramiding traits that aregenetically tightly linked. In the absence of accurate markers, limitedrecombination forces breeders to enlarge segregating populations forprogeny screens. Moreover, phenotypic evaluation is time-consuming,resource-intensive and not reproducible in every environment,particularly for traits like disease resistance. The markers provided bythe invention offer an effective alternative and therefore represent asignificant advance in the art.

Many desirable traits that are successfully introduced throughintrogression can also be introduced directly into a plant by the use ofmolecular techniques. One aspect of the invention includes plants with agenome that has been changed by any method using site-specific genomemodification techniques. Techniques of site-specific genome modificationinclude the use of enzymes such as, endonucleases, recombinases,transposases, helicases and any combination thereof. In one aspect, anendonuclease is selected from a meganuclease, a zinc-finger nuclease(ZFN), a transcription activator-like effector nucleases (TALEN), anArgonaute, and an RNA-guided nuclease, such as a CRISPR associatednuclease.

In another aspect, the endonuclease is a dCas9-recombinase fusionprotein. As used herein, a “dCas9” refers to a Cas9 endonuclease proteinwith one or more amino acid mutations that result in a Cas9 proteinwithout endonuclease activity, but retaining RNA-guided site-specificDNA binding. As used herein, a “dCas9-recombinase fusion protein” is adCas9 with a protein fused to the dCas9 in such a manner that therecombinase is catalytically active on the DNA.

Non-limiting examples of recombinase include a tyrosine recombinaseattached to a DNA recognition motif provided herein is selected from thegroup consisting of a Cre recombinase, a Gin recombinase a Flprecombinase, and a Tnp1 recombinase. In an aspect, a Cre recombinase ora Gin recombinase provided herein is tethered to a zinc-fingerDNA-binding domain, or a TALE DNA-binding domain, or a Cas9 nuclease. Inanother aspect, a serine recombinase attached to a DNA recognition motifprovided herein is selected from the group consisting of a PhiC31integrase, an R4 integrase, and a TP-901 integrase. In another aspect, aDNA transposase attached to a DNA binding domain provided herein isselected from the group consisting of a TALE-piggyBac and TALE-Mutator.

Site-specific genome modification enzymes, induce a genome modificationsuch as a double-stranded DNA break (DSB) or single-strand DNA break atthe target site of a genomic sequence that is then repaired by thenatural processes of homologous recombination (HR) or non-homologousend-joining (NHEJ). Sequence modifications then occur at the cleavedsites, which can include deletions or insertions that result in genedisruption in the case of NHEJ, or integration of exogenous sequences byhomologous recombination.

Another aspect of the invention includes transgenic plant cells,transgenic plant tissues, transgenic plants, and transgenic seeds thatcomprise the recombinant DNA molecules and engineered proteins providedby the invention. Plants comprising the recombinant DNA molecules andengineered proteins, or plants produced from the cells, tissues orseeds, have curds or heads that exhibit increased resistance to downymildew. Suitable methods for transformation of host plant cells for usewith the current disclosure include virtually any method by which DNAcan be introduced into a cell (for example, where a recombinant DNAconstruct is stably integrated into a plant chromosome) and are wellknown in the art. An exemplary and widely utilized method forintroducing a recombinant DNA construct into plants is the Agrobacteriumtransformation system, which is well known to those of skill in the art.Another exemplary method for introducing a recombinant DNA constructinto plants is insertion of a recombinant DNA construct into a plantgenome at a pre-determined site by methods of site-directed integration.Transgenic plants can be regenerated from a transformed plant cell bythe methods of plant cell culture. A transgenic plant homozygous withrespect to a transgene (that is, two allelic copies of the transgene)can be obtained by self-pollinating (selfing) a transgenic plant thatcontains a single transgene allele with itself, for example an R0 plant,to produce R1 seed. One fourth of the R1 seed produced will behomozygous with respect to the transgene. Plants grown from germinatingR1 seed can be tested for zygosity, using a SNP assay, DNA sequencing,or a thermal amplification assay that allows for the distinction betweenheterozygotes and homozygotes, referred to as a zygosity assay.

V. Molecular Assisted Breeding Techniques

Genetic markers that can be used in the practice of the presentinvention include, but are not limited to, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),simple sequence repeats (SSRs), simple sequence length polymorphisms(SSLPs), single nucleotide polymorphisms (SNPs), insertion/deletionpolymorphisms (Indels), variable number tandem repeats (VNTRs), andrandom amplified polymorphic DNA (RAPD), isozymes, and other markersknown to those skilled in the art. Vegetable breeders use molecularmarkers to interrogate a crop's genome and classify material based ongenetic, rather than phenotypic, differences. Advanced markertechnologies are based on genome sequences, the nucleotide order ofdistinct, polymorphic genotypes within a species. Such platforms enableselection for horticultural traits with markers linked to favorablealleles, in addition to the organization of germplasm using markersrandomly distributed throughout the genome. In the past, a prioriknowledge of the genome lacked for major vegetable crops that now havebeen sequenced. Scientists exploited sequence homology, rather thanknown polymorphisms, to develop marker platforms. Man-made DNA moleculesare used to prime replication of genome fragments when hybridizedpair-wise in the presence of a DNA polymerase enzyme. This synthesis,regulated by thermal cycling conditions that control hybridization andreplication of DNA strands in the polymerase chain reaction (PCR) toamplify DNA fragments of a length dependent on the distance between eachprimer pair. These fragments are then detected as markers and commonlyknown examples include AFLP and RAPD. A third technique, RFLP does notinclude a DNA amplification step. Amplified fragment length polymorphism(AFLP) technology reduces the complexity of the genome. First, throughdigestive enzymes cleaving DNA strands in a sequence-specific manner.Fragments are then selected for their size and finally replicated usingselective oligonucleotides, each homologous to a subset of genomefragments. As a result, AFLP technology consistently amplifies DNAfragments across genotypes, experiments and laboratories.

Polymorphisms comprising as little as a single nucleotide change can beassayed in a number of ways. For example, detection can be made byelectrophoretic techniques including a single strand conformationalpolymorphism (Orita, et al., Genomics 8:271-278, 1989), denaturinggradient gel electrophoresis (Myers, EP 0273085), or cleavage fragmentlength polymorphisms (Life Technologies, Inc., Gaithersburg, Md.), butthe widespread availability of DNA sequencing often makes it easier tosimply sequence amplified products directly. Once the polymorphicsequence difference is known, rapid assays can be designed for progenytesting, typically involving some version of PCR amplification ofspecific alleles (PASA; Sommer, et al., Biotechniques 12:82-87, 1992),or PCR amplification of multiple specific alleles (PAMSA; Dutton andSommer, Biotechniques 11:700-702, 1991).

Polymorphic markers serve as useful tools for assaying plants fordetermining the degree of identity of lines or varieties (U.S. Pat. No.6,207,367). These markers form the basis for determining associationswith phenotypes and can be used to drive genetic gain. In certainembodiments of methods of the invention, polymorphic nucleic acids canbe used to detect in a Brassica oleracea plant a genotype associatedwith disease resistance, identify a Brassica oleracea plant with agenotype associated with disease resistance, and to select a Brassicaoleracea plant with a genotype associated with disease resistance. Incertain embodiments of methods of the invention, polymorphic nucleicacids can be used to produce a Brassica oleracea plant that comprises inits genome an introgressed locus associated with disease resistance. Incertain embodiments of the invention, polymorphic nucleic acids can beused to breed progeny Brassica oleracea plants comprising a locusassociated with disease resistance.

Genetic markers may include “dominant” or “codominant” markers.“Codominant” markers reveal the presence of two or more alleles (two perdiploid individual). “Dominant” markers reveal the presence of only asingle allele. Markers are preferably inherited in codominant fashion sothat the presence of both alleles at a diploid locus, or multiplealleles in triploid or tetraploid loci, are readily detectable, and theyare free of environmental variation, i.e., their heritability is 1. Amarker genotype typically comprises two marker alleles at each locus ina diploid organism. The marker allelic composition of each locus can beeither homozygous or heterozygous. Homozygosity is a condition whereboth alleles at a locus are characterized by the same nucleotidesequence. Heterozygosity refers to different conditions of the allele ata locus.

Nucleic acid-based analyses for determining the presence or absence ofthe genetic polymorphism (i.e., for genotyping) can be used in breedingprograms for identification, selection, introgression, and the like. Awide variety of genetic markers for the analysis of geneticpolymorphisms are available and known to those of skill in the art. Theanalysis may be used to select for genes, portions of genes, QTL,alleles, or genomic regions that comprise or are linked to a geneticmarker that is linked to or associated with disease resistance inBrassica oleracea plants.

As used herein, nucleic acid analysis methods include, but are notlimited to, PCR-based detection methods (for example, TaqMan assays),microarray methods, mass spectrometry-based methods and/or nucleic acidsequencing methods, including whole genome sequencing. In certainembodiments, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

One method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al., Cold Spring Harbor Symp. Quant. Biol.51:263-273, 1986; European Patent 50,424; European Patent 84,796;European Patent 258,017; European Patent 237,362; European Patent201,184; U.S. Pat. Nos. 4,683,202; 4,582,788; and 4,683,194), usingprimer pairs that are capable of hybridizing to the proximal sequencesthat define a polymorphism in its double-stranded form. Methods fortyping DNA based on mass spectrometry can also be used. Such methods aredisclosed in U.S. Pat. Nos. 6,613,509 and 6,503,710, and referencesfound therein.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039;7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of whichare incorporated herein by reference in their entirety. However, thecompositions and methods of the present invention can be used inconjunction with any polymorphism typing method to type polymorphisms ingenomic DNA samples. These genomic DNA samples used include but are notlimited to, genomic DNA isolated directly from a plant, cloned genomicDNA, or amplified genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods, for example as disclosed in U.S. Pat. No. 5,800,944 wheresequence of interest is amplified and hybridized to probes followed byligation to detect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz, et al., Genome Res. 13:513-523, 2003; Cui, et al.,Bioinformatics 21:3852-3858, 2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening ofa plurality of polymorphisms. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283.

In another method for detecting polymorphisms, SNPs and Indels can bedetected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe having a 5′ fluorescentreporter dye and a 3′ quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle DNA polymerase with 5′ 4 3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

In another embodiment, a locus or loci of interest can be directlysequenced using nucleic acid sequencing technologies. Methods fornucleic acid sequencing are known in the art and include technologiesprovided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience(Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-CORBiosciences (Lincoln, Nebr.), NimbleGen Systems (Madison, Wis.),Illumina (San Diego, Calif.), and VisiGen Biotechnologies (Houston,Tex.). Such nucleic acid sequencing technologies comprise formats suchas parallel bead arrays, sequencing by ligation, capillaryelectrophoresis, electronic microchips, “biochips,” microarrays,parallel microchips, and single-molecule arrays.

Definitions

The following definitions are provided to better define the presentinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which Brassica oleraceaplants can be regenerated, plant calli, plant clumps and plant cellsthat are intact in plants or parts of plants such as pollen, flowers,curds, heads, seeds, leaves, stems, and the like.

As used herein, the term “population” means a genetically heterogeneouscollection of plants that share a common parental derivation.

As used herein, the terms “variety” and “cultivar” mean a group ofsimilar plants that by their genetic pedigrees and performance can beidentified from other varieties within the same species.

As used herein, an “allele” refers to one of two or more alternativeforms of a genomic sequence at a given locus on a chromosome.

A “Quantitative Trait Locus (QTL)” is a chromosomal location thatencodes for at least a first allele that affects the expressivity of aphenotype.

As used herein, a “marker” means a detectable characteristic that can beused to discriminate between organisms. Examples of such characteristicsinclude, but are not limited to, genetic markers, biochemical markers,metabolites, morphological characteristics, and agronomiccharacteristics.

As used herein, the term “phenotype” means the detectablecharacteristics of a cell or organism that can be influenced by geneexpression.

As used herein, the term “genotype” means the specific allelic makeup ofa plant.

As used herein, “elite” or “cultivated” variety means any plant orvariety that has resulted from breeding and selection for superioragronomic performance. An “elite plant” refers to a plant belonging toan elite variety. Numerous elite varieties are available and known tothose of skill in the art of Brassica oleracea breeding. An “elitepopulation” is an assortment of elite individuals or lines that can beused to represent the state of the art in terms of agronomicallysuperior genotypes of a given crop species, such as a Brassica oleracealine. Similarly, an “elite germplasm” or elite strain of germplasm is anagronomically superior germplasm.

As used herein, the term “introgressed,” when used in reference to agenetic locus, refers to a genetic locus that has been introduced into anew genetic background, such as through backcrossing. Introgression of agenetic locus can be achieved through plant breeding methods and/or bymolecular genetic methods. Such molecular genetic methods include, butare not limited to, marker assisted selection.

As used herein, the terms “recombinant” or “recombined” in the contextof a chromosomal segment refer to recombinant DNA sequences comprisingone or more genetic loci in a configuration in which they are not foundin nature, for example as a result of a recombination event betweenhomologous chromosomes during meiosis.

As used herein, the term “linked,” when used in the context of nucleicacid markers and/or genomic regions, means that the markers and/orgenomic regions are located on the same linkage group or chromosome suchthat they tend to segregate together at meiosis.

As used herein, “resistance locus” means a locus associated withresistance or tolerance to disease. For instance, a resistance locusaccording to the present invention may, in one embodiment, controlresistance or susceptibility of plant curds or heads to downy mildew.

As used herein, “resistance allele” means the nucleic acid sequenceassociated with resistance or tolerance to disease.

As used herein “resistance” or “improved resistance” in a plant todisease conditions is an indication that the plant is less affected bydisease conditions with respect to yield, survivability and/or otherrelevant agronomic measures, compared to a less resistant, more“susceptible” plant. Resistance is a relative term, indicating that a“resistant” plant survives and/or produces better yields in diseaseconditions compared to a different (less resistant) plant grown insimilar disease conditions. As used in the art, disease “tolerance” issometimes used interchangeably with disease “resistance.” One of skillwill appreciate that plant resistance to disease conditions varieswidely, and can represent a spectrum of more-resistant or less-resistantphenotypes. However, by simple observation, one of skill can generallydetermine the relative resistance or susceptibility of different plants,plant lines or plant families under disease conditions, and furthermore,will also recognize the phenotypic gradations of “resistant.”

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more,”unless specifically noted. The terms “comprise,” “have” and “include”are open-ended linking verbs. Any forms or tenses of one or more ofthese verbs, such as “comprises,” “comprising,” “has,” “having,”“includes” and “including,” are also open-ended. For example, any methodthat “comprises,” “has” or “includes” one or more steps is not limitedto possessing only those one or more steps and also covers otherunlisted steps. Similarly, any plant that “comprises,” “has” or“includes” one or more traits is not limited to possessing only thoseone or more traits and covers other unlisted traits.

VI. Deposit Information

A deposit was made of at least 2500 seeds of cauliflower (Brassicaoleracea) strain MYCOCLP, which comprises the two downy mildewresistance QTLs on chromosome 3, as described herein. The deposit wasmade with the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110-2209 USA. The deposit is assigned ATCCAccession No. PTA-124338, and the date of deposit was Jul. 28, 2017.Access to the deposit will be available during the pendency of theapplication to persons entitled thereto upon request. The deposit willbe maintained in the ATCC Depository, which is a public depository, fora period of 30 years, or 5 years after the most recent request, or forthe enforceable life of the patent, whichever is longer, and will bereplaced if nonviable during that period. Applicant does not waive anyinfringement of their rights granted under this patent or any other formof variety protection, including the Plant Variety Protection Act (7U.S.C. 2321 et seq.).

Example 1 Identification of Downy Mildew Resistance Alleles and Mapping

The resistant source MYCOCLP has been deposited with the ATCC andassigned Accession No. PTA-124338. MycoCLP was crossed with downy mildewsensitive brilliant white cauliflower line BSCLPN to create a mappingpopulation. The resulting F1 plants were used to develop a doubledhaploid population. In total, 198 first generation doubled haploid (DH)lines were developed and used for trial evaluation and genotype mappingtogether with the parental lines as sensitive and resistant controls.

The downy mildew resistance for this mapping population was determinedin field locations. Each trial contained 3 replicates with 10 plants foreach DH line in each replicate. In these trials, natural downy mildewisolates were relied on to infect the plants. Trials were sown in a timestaggered manner in order to anticipate variable natural infectionrates, but also to accommodate different rates of heading between thedifferent lines. In this mapping population a difference in headmaturation of about one month was observed between the different linesdepending on environmental conditions. In a first experiment, sixstaggered trials were performed with six sowing dates over a six weekperiod in April and May and six planting dates over a six week period inJune. Evaluations for this experiment occurred from August throughNovember. In a second experiment, trials were planted at the same timein two locations, with three trials in each location. For these trials,the materials were planted with three sowing dates over a three weekperiod in April and May and three respective planting dates over a threeweek period in June. These trials were evaluated from August throughNovember.

To determine the rate of downy mildew infection, mature curds wereevaluated one week after a grower would normally harvest the crops,which is between three and four months after planting. The curds wereharvested and sliced open several times to determine the level of downymildew present. Subsequently, each plant was given a score of 1 (nosymptoms), 5 (some symptoms), or 9 (multiple infection sites and/or >1/3of the curd infected). For each line, the results from the twoexperimental trials were combined and the downy mildew score wassummarized into the least square mean.

Each DH line was genotyped and QTL analyses were undertaken with MapQTL5using interval mapping at a 1 cM mapping step size. Significancethresholds were determined by permutation tests with 1000 permutationseach and a threshold of p=0.05. The QTL mapping analysis identified twoQTLs on chromosome 3 that together explain 48.1% of the phenotypicvariation around downy mildew resistance in the cauliflower curds (FIG.1).

To reduce the size of the first QTL (between markers M13 to M20),recombinants were identified using the flanking markers M13 and M20 inthe F2 generation of a cross between the same parents as used for theQTL mapping. These recombinants were made homozygous for the recombinantbreak points in the F3 generation. The F4 generation recombinantfamilies were planted in trials for downy mildew resistance. The trialwas replicated in six staggered sowings and plantings and was plantedacross two locations. By aligning the least square mean level ofresistance for each of these recombinant families to their respectiverecombination breakpoints between M13 and M20, the inventors identifiedthat the region between markers M19 and M20 provided the resistanceconveyed by the mapped QTL.

The sequences for the markers described herein are shown in Table 1.

TABLE 1 SNP Position on Position Marker Position in Proprietary SNP v2.1Size Marker SNP Marker QTL Map (cM) (bp) (bp) (bp) Change Sequence M13 178 Unknown 121 61 T/C SEQ ID NO: 1 M19 1 82 15,890,285 121 61 G/A SEQ IDNO: 2 M20 1 93 10,184,762 121 61 T/C SEQ ID NO: 3 M31 2 127 3,226,172121 61 T/A SEQ ID NO: 4 M33 2 128 3,178,026 121 61 G/A SEQ ID NO: 5 M342 129 2,874,663 121 61 T/C SEQ ID NO: 6 M35 2 131 2,354,342 121 61 A/GSEQ ID NO: 7 M36 2 133 2,168,486 121 61 C/T SEQ ID NO: 8 M37 2 1332,212,440 118 58 A/C SEQ ID NO: 9 M38 2 133 1,973,175 121 61 C/A  SEQ IDNO: 10 M39 2 134 1,391,141 121 61 T/G  SEQ ID NO: 11 M40 2 135 1,932,167121 61 A/G  SEQ ID NO: 12 M41 2 135 2,091,771 84 61 A/G  SEQ ID NO: 13M42 2 137 1,220,020 94 61 A/G  SEQ ID NO: 14 M43 2 137 1,219,392 121 61A/G  SEQ ID NO: 15 M44 2 137 1,221,810 121 61 C/T  SEQ ID NO: 16

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A Brassica oleracea plant of a cultivated variety, the plantcomprising a first introgressed allele or a second introgressed alleleon chromosome 3, wherein said first introgressed allele or said secondintrogressed allele confers to a curd or head of said plant increasedresistance to downy mildew compared to a plant not comprising said firstintrogressed allele or said second introgressed allele.
 2. The Brassicaoleracea plant of claim 1, wherein said plant comprises a firstintrogressed allele and a second introgressed allele on chromosome 3,wherein said first introgressed allele and said second introgressedallele confers to a curd or head of said plant increased resistance todowny mildew compared to a plant not comprising said alleles.
 3. TheBrassica oleracea plant of claim 1, wherein a sample of seed comprisingsaid first introgressed allele and said second introgressed allele wasdeposited under ATCC Accession Number PTA-124338.
 4. The Brassicaoleracea plant of claim 1, wherein said first introgressed allele isflanked in the genome of said plant by marker locus M19 (SEQ ID NO:2)and marker locus M20 (SEQ ID NO:3) on chromosome 3 or wherein saidsecond introgressed allele is flanked in the genome of said plant bymarker locus M31 (SEQ ID NO:4) and marker locus M44 (SEQ ID NO:16) onchromosome
 3. 5. (canceled)
 6. The Brassica oleracea plant of claim 1,wherein said Brassica oleracea plant is a broccoli, cauliflower,sprouting broccoli, Brussels sprouts, white cabbage, red cabbage, savoycabbage, curly kale cabbage, turnip cabbage or Portuguese cabbage plant.7. The Brassica oleracea plant of claim 1, wherein the plant ishomozygous for said first introgressed allele or said secondintrogressed allele.
 8. A seed that produces the Brassica oleracea plantof claim
 1. 9. A plant part of the Brassica oleracea plant of claim 1.10. The plant part of claim 9, wherein said plant part is a cell, aseed, a root, a stem, a leaf, a fruit, a flower, a curd, a head orpollen.
 11. An introgression fragment comprising a first chromosomalsegment on chromosome 3 from Brassica oleracea MYCOCLP flanked by markerM19 (SEQ ID NO:2) and marker M20 (SEQ ID NO:3) and a second chromosomalsegment on chromosome 3 from Brassica oleracea MYCOCLP flanked by markerM31 (SEQ ID NO:4) and marker M44 (SEQ ID NO:16).
 12. The introgressionfragment of claim 11, wherein said fragment confers increased resistanceto downy mildew.
 13. The introgression fragment of claim 12, wherein asample of seed comprising said first chromosomal segment and said secondchromosomal segment was deposited under ATCC Accession NumberPTA-124338.
 14. A method for producing a cultivated variety of aBrassica oleracea plant with a curd or head having improved resistanceto downy mildew, comprising introgressing into said plant variety afirst chromosomal segment or a second chromosomal segment from Brassicaoleracea MYCOCLP chromosome 3 that confers improved resistance to downymildew relative to a plant lacking said introgression.
 15. The method ofclaim 14, wherein said introgressing comprises: a) crossing a plantcomprising said first or second chromosomal segment with itself or witha second Brassica oleracea plant of a different genotype to produce oneor more progeny plants; and b) selecting a progeny plant comprising saidchromosomal segment.
 16. The method of claim 15, wherein selecting aprogeny plant comprises detecting at least a first allele flanked bymarker M19 (SEQ ID NO:2) and marker M20 (SEQ ID NO:3) or a second alleleflanked by marker M31 (SEQ ID NO:4) and marker M44 (SEQ ID NO:16). 17.The method of claim 14, wherein said plant variety is a broccoli,cauliflower, sprouting broccoli, Brussels sprouts, white cabbage, redcabbage, savoy cabbage, curly kale cabbage, turnip cabbage or Portuguesecabbage plant variety.
 18. The method of claim 15, wherein the progenyplant is an F2-F6 progeny plant.
 19. The method of claim 15, whereinsaid crossing comprises backcrossing, wherein said crossing comprisesfrom 2-7 generations of backcrosses or wherein said crossing comprisesmarker-assisted selection.
 20. (canceled)
 21. (canceled)
 22. The methodof claim 14, wherein a sample of seed comprising said first and secondchromosomal segment was deposited under ATCC Accession NumberPTA-124338.
 23. A Brassica oleracea plant produced by the method ofclaim
 14. 24. A method of producing food or feed comprising obtaining aplant according to claim 1, or a part thereof, and producing said foodor feed from said plant or part thereof.
 25. A method of selecting aBrassica oleracea plant exhibiting resistance downy mildew, comprising:a) crossing the Brassica oleracea plant of claim 1 with itself or with asecond Brassica oleracea plant of a different genotype to produce one ormore progeny plants; and b) selecting a progeny plant comprising saidfirst or second introgressed allele.
 26. (canceled)
 27. The method ofclaim 26, wherein selecting said progeny plant comprises identifying agenetic marker genetically linked to said first or second introgressedallele or wherein selecting said progeny plant comprises: a) identifyinga genetic marker within or genetically linked to a genomic regionbetween marker locus M19 (SEQ ID NO:2) and marker locus M20 (SEQ IDNO:3) on chromosome 3; or b) identifying a genetic marker within orgenetically linked to a genomic region between marker locus M31 (SEQ IDNO:4) and marker locus M44 (SEQ ID NO:16) on chromosome
 3. 28. Themethod of claim 27, wherein selecting a progeny plant further comprisesdetecting at least one polymorphism at a locus selected from the groupconsisting of marker locus M33 (SEQ ID NO:5), marker locus M34 (SEQ IDNO:6), marker locus M35 (SEQ ID NO:7), marker locus M36 (SEQ ID NO:8),marker locus M37 (SEQ ID NO:9), marker locus M38 (SEQ ID NO:10), markerlocus M39 (SEQ ID NO:11), marker locus M40 (SEQ ID NO:12), marker locusM41 (SEQ ID NO:13), marker locus M42 (SEQ ID NO:14), and marker locusM43 (SEQ ID NO:15).
 29. The method of claim 25, wherein said progenyplant is an F2-F6 progeny plant.
 30. The method of claim 25, whereinproducing said progeny plant comprises backcrossing.
 31. (canceled) 32.A Brassica oleracea plant obtainable by the method of claim 25.